Antifungal Effect of Probiotic Lactobacillus casei on Drug-Resistant Oral Candida albicans Isolated from Patients with Hematological Malignancy: an in vitro Study

Statement of the Problem: Candida albicans (C. albicans) is recognized as the most common opportunistic pathogen in patients with an impaired immune system, and due to the frequent use of antifungal medicine, a variety of drug-resistant species are developing. Probiotics are a part of the human microbiome and natural competitors of Candida by producing lactic acid, low pH, and other secreted metabolites. The role of probiotics in preventing fungal infections has always been discussed. Purpose: This study aimed to investigate the antifungal effect of Lactobacillus casei (L. casei) on fluconazole- and amphotericin B-resistant C. albicans species isolated from the oral cavity of acute myeloid leukemia patients. Materials and Method: In this experimental study, eight strains of fluconazole- and amphotericin B-resistant C. albicans were used. The antifungal effects of probiotic L. casei and nystatin were measured by the co-aggregation method 1, 2, and 4 h after beginning the study. After each hour of exposure, C. albicans and L. casei colonies were counted. Results: L. casei had a significant ability to aggregate with both fluconazole- and amphotericin B-resistant C. albicans in all designated intervals, which increased with time. In the first hour of the study, no significant difference was observed between the effects of L. casei on the two drug-resistant strains. However, as time passed, it had a more significant antifungal effect on fluconazole, compared to amphotericin B resistant species (p Value<0.001). Cell counts showed that the number of fungal cells decreased significantly as time passed (p< 0.001). Conclusion: L. casei had a significant ability to aggregate with both drug-resistant C. albicans species and showed higher antifungal activity on fluconazole-resistant than amphotericin B-resistant species.


Introduction
Candida albicans (C.albicans) is recognized as the most common opportunistic pathogen in impaired immune system patients, and its prevalence is increasing with the growing number of immunocompromised people [1][2].It is clear that patients with a weakened immune system, due to chemotherapy and radiotherapy for cancer treatment, are highly prone to oral candidiasis.The quality of these patients' life is completely affected by fungal infections, and they may experience severe, invasive, and even life-threatening side effects following the entry of Candida species into their bloodstream (candidemia) [1][2][3][4].The colonization of C. albicans in immune-deficient patients, especially in hematological malignancies, can affect the final treatment results [5].
Currently, following the frequent, long-term, and proph-ylactic use of antifungal medications in patients with candidiasis, the variety of drug-resistant Candida strains is increasing.Therefore, finding an alternative way to control and prevent this group of fungal infections is suggested [4,6].Some studies [7][8][9][10] show that Candida species colonization is reduced following the consumption of foods containing probiotics [7].Lactobacillus species are among probiotics that inhibit the formation and growth of Candida biofilm and reduce the symptoms of infection.The role of probiotics in preventing infectivity is not fully understood, and it is currently hypothesized that probiotics inhibit the growth of fungal cells by competing for food, occupying the sites required for Candida attachment, and producing antimicrobial agents.Probiotics can also improve the mucosal immune system [8][9][10].Therefore, this study aims to investigate the role of probiotic Lactobacillus casei (L.casei) in the prevention of fungal lesions in the form of co-aggregation.

L. casei and culture conditions
In this in vitro experimental study, the probiotic L. casei (PTCC number 1608) was used, provided by the Iranian Research Organization for Science and Technology.
The L. casei was cultured under anaerobic conditions in Man Rogosaf Sharpe (MRS, SIGMA, USA) medium at 37˚C for 24 h.The isolated L. casei colonies were transferred to the MRS medium (5 ml) and incubated at 37˚C for 48 h.After the incubation, L. casei was stocked at 20˚C in glycerol.1 ml of L. casei was relocated to 5 ml of MRS broth medium for recultivation.Finally, Lcysteine (50 μl) was added.The microtubes were located in a shaker incubator (16B; KTG laboratory equipment) at 37˚C for 20 h [8].

C. albicans and culture conditions
A microbiologist isolated the C. albicans from the oral cavity of patients with acute myeloid leukemia admitted to the Taleghani Hospital of Shahid Beheshti University (Tehran, Iran).These patients did not show any im- nystatin and amphotericin B resistant C. albicans [8].

Co-aggregation of C. albicans and L. casei
The co-aggregation was assessed by a spectrophotometer (PD-303; Apple model).At first, the mixture of L. casei and C. albicans was incubated for 1, 2, and 4 h.
Then, the co-aggregation ratio was determined consistent with the methodology proposed by Salari et al. [8] Initially, the detached colonies of 24-h L. casei were moved to 5 ml MRS broth medium and were incubated in a shaker incubator (84 RPM; 24 h; 37˚C) in anaerobic carriage.The C. albicans was isolated from SDA and cultured in SDB at 37˚C for 24 h.Then, the microtubes containing L. casei and C. albicans were centrifuged (855 RPM; 10 min, 25˚C).The supernatant was removed and the sediment was washed three times in phosphate buffer saline (PBS, SIGMA, USA) and suspended in 10 mmol/L PBS (at pH=7).The optical absorbance was measured by a spectrophotometer at the specific wavelength of 600 (OD600 nm) nanometers equivalent to the McFarland standard (10 8 CFU/mL for Lactobacilli and 10 6 CFU/mL for Candida species).In total, 1ml of each L. casei and 1 ml of C. albicans were totally mixed and incubated in a shaker incubator (100 rpm; 37˚C; for 1, 2, and 4 h).Then, the optical density (OD) measurement was performed with spectrophotometer at OD600 nm.Each experiment was repeated three times [8].
The percentage of co-aggregation was calculated ba-sed on the following equation:

Co-aggregation percentage between L. casei and two drugresistant C. albicans in 1, 2, and 4 hours of study
The normality of data distribution was checked by Kolmogorov-Smirnov test.Based on the test results, the distribution of all data was normal.One-way ANOVA results showed that in all three times (1, 2, and 4 h), L. casei had a significant ability to aggregate with drug-resistant C. albicans, which increased with time (p< 0.001).

First hour of the study
There was no significant difference between the co-aggregation degree of L.casei with fluconazole-resistant and amphotericin B-resistant C. albicans, and the antifungal effect was almost similar.At the same time, compared to nystatin, L. casei had a significantly lower antifungal effect on amphotericin B resistant species (p< 0.001).On the other hand, nystatin had a slightly more antifungal effect on fluconazole-resistant species when comp-ared to L. casei, but the difference was not significant (Figure 1).

Second hour of the study
Herein, a significant difference was monitored between  albicans (p< 0.001).Moreover, L. casei had greater coaggregation ability with fluconazole-resistant species than amphotericin B-resistant strains.At this time, no significant difference was observed between the antifungal results of L. casei and nystatin on amphotericin B and fluconazole-resistant species.However, the findings revealed that in amphotericin B-resistant species, the effect of nystatin was slightly better, and in fluconazoleresistant species, L. casei had an antifungal effect similar to that of nystatin (Figure 2).

Fourth hour of the study
In the fourth hour, L. casei had a significantly higher coaggregation rate with fluconazole-resistant than amphotericin B-resistant species (p< 0.001).Furthermore, compared to nystatin, L. casei had a significantly greater   was better than all groups and had a significant antifungal effect, in comparison with other groups the study (p< 0.001) (Figure 3).

Discussion
Due to the increasing prevalence of oral candidiasis in immunodeficient patients and the development of drugresistant Candida species, the use of prophylactic components, such as probiotics, for preventing fungal infections has always been proposed [11][12][13][14].The role of probiotics in this field is generally recognized, and various studies have revealed its role in reducing the signs and symptoms of oral candidiasis [15][16].Several experiences have revealed antifungal effects of different probiotics on oral C. albicans [8][9][17][18].This study tested the antifungal effect of probiotic L. casei on fluconazole-and amphotericin B-resistant C. albicans by evaluating the degree of co-aggregation and the colony counting method.In the study of Salari et al. [8], the ability of co-aggregation was seen between L .acidophilus and L .plantarum with all tested Candida strains, and their co-aggregation percentage increased over time.
The highest co-aggregation rate was observed between C. krusei and two probiotics, which was lower than that of C. albicans.In a study by Jørgensen et al. [19], L. reuteri with C. tropicalis and C. krusei had the greatest co-aggregation ratio.Chew et al. [20] also stated that L. reuteri showed a mainly greater co-aggregation ratio versus C. glabrata species, in comparison with L. rhamnosus.Therefore, it appears that co-aggregation levels are exclusive for each species of Lactobacilli [8].In the next part of this study, colony counting was performed 1, 2, and 4 hours after the study began.Based on counti-  [16,22].Previous study has also shown that L. casei has the ability to produce Interleukin-12, which stimulates natural killer cells (first line of defense) to respond against the infection and their proper function plays an important role in the prognosis of the disease [18].In a study by Villena et al [9], L. casei was found to prevent the damage caused by inflammatory responses by producing interleukin-10 (IL-10).The IL-10 is a potent anti-inflammatory regulatory cytokine that has a beneficial effect on the host's response against fungal infections.In the early stages of infection, cytokines can interfere with the antifungal function of phagocytes and the production of other proinflammatory cytokines.
However, in the later stages of infection, the increase in IL-10 production may help relieve the inflammatory response.Therefore, the use of this probiotic as a sup-plement with the mechanism of increasing IL-10 prevents the damage caused by inflammatory responses [9].The difficulty of Lactobacillus bacteria growth in culture medium and the sensitivity of probiotics to environmental conditions were some of the possible limitations.A novel feature of our survey was the comparison between the antifungal effects of L. casei on fluconazole-and amphotericin B-resistant C. albicans.These species were isolated from the oral cavity of AML patients.The incidence of oral candidiasis is associated immunity reduction followed by a chemotherapy regimen in patients with hematological malignancies.

Conclusion
The findings revealed that L. casei had a significant ability to co-aggregate with both drug-resistant C. albicans species and showed higher antifungal activity on fluconazole-resistant strains than amphotericin B-resistant species.Further investigations are recommended for assessing the antifungal properties of other Lactobacillus species on drug-resistant C. albicans and recognizing the precise mechanisms of their action, as well as performing antifungal susceptibility patterns in models of infected animal.
provement in oral lesions following the administration of fluconazole and amphotericin B, either clinically or microscopically (in terms of colony count, cell numbers, and fungal wall destruction).Sampling was done with two swabs prepared from the oral lesions caused by Candida species.They were placed in 1 cc of distilled water and transferred to the laboratory.A direct slide was prepared from one swab and the second swab was prepared on Saburo Dextrose Agar (SDA, SIGMA, US-A) medium.Minimum inhibitory concentration results were determined according to the CLSI-M27-S3 (Clinical & Laboratory Standards Institute) and showed that the strains were resistant to fluconazole and amphotericin B (13).Candida species were stored at -70˚C in Tryptic Soy Broth (SIGMA, USA).To start the study, Candida species were cultured in SDA and transferred to Saburo Dextrose Broth (SDB) for 24 h at 37˚C.Eight strains of fluconazole-and amphotericin B-resistant C. albicans were used, and the antifungal effects of L. casei (study groups) were measured, in comparison with nystatin (control groups), by the co-aggregation method 1, 2, and 4 h after the study began.The study groups included (1) L. casei and fluconazole-resistant C. albicans, (2) L. casei and amphotericin B resistant C. albicans, (3) nystatin and fluconazole-resistant C. albicans, and (4) OD 0 : the absorption amount of the complex suspension of L. casei with C. albicans at the beginning of the trail (0 h). OD h : the absorption amount of the complex solutions at various times L. casei and C. albicans cell counts To evaluate the cell number of C.albicans and L.casei 1, 2, and 4h after the study began (after measuring the absorbance of the solution by the spectrophotometer OD60-0nm), some of the solution containing L.casei and C.albicans was removed with an inoculation needle and transferred to the culture medium.After 24h, the number of cells was determined by counting the colonies of each species separately and calculating the number of cells by calculating each colony in a certain coefficient of dilution (coefficient of dilution was prepared based on the diameter of the needle loop).Small colonies were belonged to L.casei and larger colonies revealed C.albicans.Data analysis Data were analyzed by the SPSS 11 software through one-way ANOVA and Tukey HSD tests.

Figure 1 :
Figure 1: Co-aggregation percentage of L. casei with drugresistant C. albicans species in comparison with Nystatin, in the first hour of the study

Figure 2 :
Figure 2: Co-aggregation percentage of L. casei with drugresistant C. albicans species in comparison to Nystatin, in the second hour of the study

Figure 3 :
Figure 3: Co-aggregation percentage of L. casei with drugresistant C. albicans species in comparison to Nystatin, in the fourth hour of the study Lactobacilli casei and drug-resistant C. albicans after 1, 2, and 4 hours of exposure Cell number of L. casei and resistant C. albicans after 1, 2, and 4 hours of exposure showed that the amount of L. casei and drug-resistant C. albicans cells decreased significantly over time (p< 0.001).It was also found that the amount of fluconazole-resistant C. albicans and L. casei cells decreased more, in comparison with L. casei and amphotericin B-resistant cells in the fourth hour of the study (p< 0.001) (Figure 4).

Figure 4 :
Figure 4: a: Smaller colonies were belonged to L. casei and larger colonies revealed C. albicans.control group, b: L. casei and amphotericin B resistant C. albicans, c: L. casei and fluconazole resistant C. albicans